Induction of gene silencing in Haemaphysalis longicornis ticks through immersion in double-stranded RNA

- Immersion of all tick stages in dsRNA dissolved in water resulted in gene silencing.
- Immersion at 15 °C for 12 h is the optimum condition to induce silencing.
- Adults immersed in dsHlfer1 had post-feeding phenotypes similar to injected adults.

The continuous emergence of tick-borne diseases and chemical acaricide-resistant tick strains necessitates the development of new and more effective control strategies. RNA interference through the injection of double-stranded RNA (dsRNA) has been a very useful tool in tick research for evaluating gene function. However, this technique can be sophisticated due to the required equipment and technique. Here we studied the feasibility of an immersion technique to induce gene silencing in Haemaphysalis longicornis ticks. We targeted the Hlfer1 gene, previously shown to be crucial in successful blood feeding and reproduction. Larval, nymphal, and adult female H. longicornis ticks were immersed in Hlfer1 or Luciferase dsRNA for control. The dsRNA dissolving medium, incubation temperature and time were varied to establish the optimum conditions. RT-PCR was performed to confirm gene silencing. It was found that immersing the ticks in dsRNA dissolved in nuclease-free water at 15 °C for 12 h resulted in clear gene silencing. The phenotypes of adult ticks immersed in dsRNA were then compared with those of adult ticks injected with dsRNA. Similar to dsRNA injection, the post-blood meal weight of ticks immersed in Hlfer1 dsRNA was significantly lower than the control group. Moreover, high post-blood meal mortality and low egg output was observed both from ticks injected with and immersed in Hlfer1 dsRNA. Our results here suggest that immersion in dsRNA can effectively induce gene silencing and not only offers an alternative method to dsRNA injection but also opens the possibility of applying dsRNA for tick control.

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